TWI607766B - Nucleic acid, medical nanoparticle(s), and pharmaceutical composition thereof - Google Patents

Description

Nucleic acid, medical nanoparticle group, and pharmaceutical composition

This invention relates to a novel nanoparticle, and more particularly to a nucleic acid, a medical nanoparticle group, and a pharmaceutical composition.

Cancer, also known as malignant tumor, is a disease in which cells are abnormally proliferated and these proliferating cells further invade other tissues/organs of the individual. According to statistics, in 2012, about 14.1 million people suffered from cancer, while nearly 8.2 million died of cancer, accounting for 14.6% of the total number of deaths in the year. Common cancers in men are lung cancer, prostate cancer, colorectal cancer and stomach cancer; common cancers in women are breast cancer, colorectal cancer, lung cancer and cervical cancer; children are most common in acute lymphocytic leukemia and brain cancer. The causes of cancer are extremely complex and diverse, such as genetic inheritance, obesity, smoking, drinking, lack of exercise, infection, radiation, eating habits, and chronic inflammation that can lead to cell cancer. In 2015, Johns Hopkins University's research also pointed out that cancer production is related to luck, which is caused by random mutations that occur when cells divide. However, the exact cause of cancer is still unclear and it has deepened the difficulty of prevention and treatment.

Many academic studies have shown that cancer cell division has the following characteristics compared to normal cells: (1) rapid metabolic rate; (2) immortalization, which can continue to divide without aging or death; (3) The gene is extremely unstable, the mutation rate is high, and it is easy to produce drug resistance; (4) It can produce a large amount of vascular endothelial growth factor. Factor, VEGF), epidermal growth factor (EGF), fibroblast growth factor (FGF) and hepatocyte growth factor (HGF) and other growth factors, and rapidly proliferate, and Promote angiogenesis; and (5) high invasive and metastatic ability. At present, the treatment of cancer is mainly based on surgical resection; in addition, depending on the characteristics of different cancers and the condition or needs of patients, medical personnel will also moderately administer chemotherapy, radiation therapy, immunotherapy or monoclonal antibody therapy. However, based on the characteristics of the aforementioned cancers, various therapies (whether administered alone or in combination) often fail to produce the desired therapeutic efficacy. In addition, most treatment methods not only poison cancer cells after administration, but also cause serious damage to normal normal cells, resulting in unavoidable side effects and health hazards. What's more, when the cancer metastasizes, no matter what method can't effectively inhibit the growth of cancer cells, it will eventually cause the cancer cells to over/extensively invade the important tissues/organs of the individual, leading to the death of the individual.

In view of the fact that cancer is still regarded as an incurable disease, various traditional therapies can not produce satisfactory effects; therefore, there is a need in the related field for a method for effectively treating cancer to alleviate pain and discomfort of patients, thereby improving Its quality of life and its longevity.

A medical nanoparticle group comprising a first core, a first outer lipid layer, a first inner lipid layer and a nucleic acid. The core contains Bio-degradable ionic precipitate (BIP). The first inner lipid layer is between the first core and the first outer lipid layer. The nucleic acid is on the surface of the core and the nucleic acid is the nucleotide sequence of SEQ ID NO: 1.

A pharmaceutical composition comprising a pharmaceutically acceptable additive and the aforementioned group of medical nanoparticles.

A nucleic acid which is a nucleotide sequence of SEQ ID NO: 1.

A pharmaceutical composition comprising the nucleic acid described above and a pharmaceutically acceptable additive.

In summary, in some embodiments, nucleic acids, medical nanoparticle groups, and pharmaceutical compositions according to the present invention may utilize nucleic acids (eg, ribonucleic acids such as small interfering ribonucleic acids, small hairpin ribonucleic acids, or microribonucleic acids). Interference) to inhibit the performance and function of the epidermal growth factor receptor, thereby inhibiting the growth of cancer cells and/or promoting cancer cell death. In some embodiments, since the photosensitizer in the medical nanoparticle group is irradiated/excited after receiving a specific light source, free radicals are released, thereby causing oxidative damage of the cancer cells. In some embodiments, the photosensitizer in the medical nanoparticle group is more useful for diagnosis. In some embodiments, the nucleic acid, the medical nanoparticle group, and the pharmaceutical composition according to the present invention can simultaneously treat the tumor with different inhibition pathways using nucleic acids and photosensitizers, in order to achieve a better therapeutic effect.

100‧‧‧ core

110‧‧‧Biodegradable ionic precipitates

400‧‧‧nucleic acid

500‧‧‧Photosensitizer

200‧‧‧ Inner fat layer

300‧‧‧ outer lipid layer

310‧‧‧Lipid-polyethylene glycol conjugate

320‧‧‧Fennelamide

[Fig. 1A] is the result of the cytotoxicity test (MTT test) described in Experimental Example 1, in which SCC4 cells were analyzed for survival by MTT assay after receiving porphyrin phototherapy.

[Fig. 1B] is the result of the MTT assay described in Experimental Example 1, in which SCC4 cells were analyzed for survival by MTT assay after receiving pyropheophorbide-phosphatidic acid treatment.

[Fig. 1C] is the result of the MTT assay described in Experimental Example 1, in which SAS cells were analyzed for survival by MTT assay after receiving porphyrin phototherapy.

[Fig. 1D] is the result of the MTT test described in Experimental Example 1, in which SAS cells are accepted After treatment with pyropheophorbide-phosphatidic acid, the survival rate was analyzed using the MTT assay.

[Fig. 2A] is a histogram described in Experimental Example 1, which utilizes porphyrin phototherapy and pyropheophorbide-phosphatidic acid to treat SCC4 cells, respectively, and then uses MTT assay to analyze cell survival results.

[Fig. 2B] is a histogram described in Experimental Example 1, which utilizes porphyrin phototherapy and pyropheophorbide-phosphatidic acid to treat SAS cells, respectively, and then uses MTT assay to analyze cell survival results.

[Fig. 3A] is a schematic view of medical nanoparticles according to an embodiment of the present invention.

[Fig. 3B] is a schematic view of a medical nanoparticle according to another embodiment of the present invention.

[Fig. 3C] is a schematic view of a medical nanoparticle according to still another embodiment of the present invention.

[Figs. 4A to 4C] are electron micrographs described in Experimental Example 2, in which the nanoparticles are coated with different concentrations of epidermal growth factor receptor small interfering ribonucleic acid (EGFR siRNA) and pyropheophorbide. - Phosphatidic acid and analysis using a transmission electron microscope.

[Figs. 5A to 5F] are electron micrographs as set forth in Experimental Example 3, in which the particles in the 5A, 5C, and 5E are the core of the nanoparticle (including the lining layer), 5B, 5D, and 5F. The particles in the figure are nanoparticle coated with a lipid layer and an outer lipid layer; the particles in Figures 5A and 5B contain EGFR siRNA, and the particles in the 5C and 5D images contain pyropheophorbide-phospholipid For the acid, the particles in Figures 5E and 5F contain both EGFR siRNA and pyropheophorbide-phosphatidic acid; all photographs are analyzed using a transmission electron microscope.

[Fig. 6] is the photo per value (cps) described in Experimental Example 5, which is obtained by injecting a nanoparticle into a mouse and using a light wave having a wavelength of 410 nm, and The photon values of the nanoparticles in the 670-690 nm and 710-730 nm wavelength sections were observed.

[Fig. 7] The daily tumor volume change of nude mice xenografted with SCC4 oral cancer cells at 0-13 days.

Although numerical values and parameters are used to define a wide range of values, the relevant values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within the acceptable standard error of the average, depending on the considerations of those of ordinary skill in the art to which the invention pertains. Except for the experimental examples, or unless otherwise explicitly stated, all ranges, quantities, values, and percentages used herein are understood (eg, to describe the amount of material used, the length of time, the temperature, the operating conditions, the quantity ratio, and the like. Are all modified by "about". Therefore, unless otherwise indicated to the contrary, the numerical parameters disclosed in the specification and the appended claims are intended to be At a minimum, these numerical parameters should be understood as the number of significant digits indicated and the values obtained by applying the general carry method.

The scientific and technical terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention pertains, unless otherwise defined herein. In addition, the singular noun used in this specification covers the plural of the noun in the case of no conflict with the context; and the plural noun of the noun is also used in the plural noun used.

In the present specification, words such as "Nucleotide sequence", "polynucleotide" or "nucleic acid" are mutually They are used interchangeably and mean a double strand of DNA, a single strand of DNA or a transcription product of DNA (eg, an RNA molecule). It is to be understood that the present invention is not related to genetic polynucleotide sequences in nature or in nature.

In the present specification, "ribonucleic acid interference (RNAi)" refers to an RNA molecule for silencing or reducing gene expression, including small interference ribonucleic acids (siRNA), small hairpins. Small hairpin ribonucleic acids (shRNA) or micro-ribonucleic acids (miRNA). Generally, these RNA molecules transcribe a silent gene by homologous to the silent gene sequence to produce sequence specificity in the animal or plant. These RNA molecules can be endogenous or exogenous, can also be integrated into a chromosome or expressed as an extrachromosomal transfection vector. In effect, these RNA molecules can completely or partially inhibit the expression of the target gene; or, by completely or partially inhibiting the function of the target gene, the effect of silence can be produced.

In the present specification, the term "therapeutic" means the application or administration of the nucleic acid, medical nanoparticle and/or pharmaceutical composition thereof according to any of the embodiments of the present invention to the symptoms and/or related symptoms of cancer. A body that achieves partial or complete alleviation, alleviation, cure, delay, inhibition, termination, and/or reduction of the occurrence of a related condition, associated symptom, course of disease, clinical marker, or a combination thereof of one or more cancers. Among them, cancer related symptoms and related symptoms include, but are not limited to, swelling, bleeding, pain, ulcers, swollen lymph nodes, cough, hemoptysis, hepatomegaly, bone pain, fracture, weight loss, loss of appetite, anemia or Its combination. The term "treatment" as used herein may also mean the administration of a nucleic acid, medical nanoparticle and/or pharmaceutical composition thereof according to any of the embodiments of the present invention to an individual having such early signs or symptoms to reduce the development of the individual. Cancer related diseases The risk of remission or related symptoms.

The term "subject" as used herein refers to a human-containing animal that is capable of receiving treatment with a nucleic acid, medical nanoparticle, and/or pharmaceutical composition of any of the embodiments of the present invention. Unless specifically stated otherwise, the term "individual" means both male and female, and can be of any age, such as a child or an adult.

In the present specification, "effective amount" means an amount of a drug sufficient to produce a desired therapeutic response. The specific effective amount depends on a number of factors, such as the particular condition being treated, the individual's physiological condition (eg, individual weight, age, or sex), the type of individual being treated (eg, rabbit, mouse, human, monkey, or race) Etc.), duration of treatment, the nature of the current therapy, if any, and the specific formulation used and the structure of the compound or derivative thereof. "Effective amount" also refers to a compound or composition whose therapeutic benefit outweighs its toxic or detrimental effects. For example, an effective amount can be expressed as the total weight of the drug (eg, in grams (g), milligrams (mg), or micrograms (μg)), or as a ratio of drug weight to body weight (in milligrams per unit) Kilograms (mg/kg)). Alternatively, an effective amount can be expressed as the concentration of the active ingredient in the pharmaceutical composition, such as molar concentration, mass concentration, volume concentration, molarity, and Mo. The mole fraction, the mass fraction, and the mixing ratio. Those of ordinary skill in the art can calculate the human equivalent dose of a drug (such as a nucleic acid, medical nanoparticle, and/or pharmaceutical composition of any of the embodiments of the present invention) based on the dose of the animal model. HED). For example, those of ordinary skill in the art can estimate the adult according to the US Food and Drug Administration (FDA). Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers to estimate the highest safe dose for human use.

In the present specification, "photodynamic therapy (PDT)" refers to the use of visible light (usually produced by non-thermal lasers) to excite a photosensitizer to treat a tumor.

In the present specification, "photodynamic diagnosis (PDD)" refers to the use of visible light (usually produced by non-thermal lasers) to excite a photosensitizer to diagnose a tumor. When the photosensitizer molecule absorbs the irradiation energy of a specific light wavelength in the ground state and jumps to the excited state, the molecule releases the energy if the molecule directly releases the energy in the excited single state state (Singlet state). The detectable fluorescence returns to the ground state, which is the principle of photodynamic diagnosis, whereby the fluorescence of the photosensitizer can be used to diagnose the location of the cancer lesion.

As used herein, the term "pharmaceutical composition" is used to encompass a composition comprising one or more active elements suitable for therapeutic use (eg, a nucleic acid or a medical na[beta] according to any of the embodiments. Rice particles).

In the present specification, "pharmaceutically acceptable excipient" means a substance which is suitable for use with a main drug without causing excessive harmful side effects (such as toxicity, irritation and allergic reaction) and having An appropriate benefit/hazard ratio. In addition, each additive must be "acceptable", i.e., compatible with the remainder of the pharmaceutical composition. The additive may be in the form of a solid, semi-solid, liquid diluent, paste or capsule.

In one embodiment, please refer to FIG. 3A, a medical nanoparticle comprising a nano particle (hereinafter referred to as a first nano particle), and the first nano particle comprises a core 100 and a foreign fat. Layer 300, an inner lipid layer 200, and nucleic acid 400. This core 100 comprises a Bio-degradable ionic precipitate (BIP) 110. The inner lipid layer 200 is located between the core 100 and the outer lipid layer 300, and the nucleic acid 400 is located on the surface of the core 100. In some embodiments, the inner lipid layer 200 can be the surface of the core 100 and the nucleic acid 400 is embedded on the inner lipid layer 200. In other words, one end of the nucleic acid 400 is located inside the core 100, and the other end is located outside the core 100.

In another embodiment, please refer to FIG. 3B, a medical nano particle comprising a nano particle (hereinafter referred to as a second nano particle), and the second nano particle comprises a core 100, an outer The lipid layer 300, an inner lipid layer 200 and a photosensitizer 500. And this core 100 contains a biodegradable ion precipitate 110. The inner lipid layer 200 is located between the core 100 and the outer lipid layer 300, and the photosensitizer 500 is located on the surface of the inner lipid layer 200 between the outer lipid layer 300 and the inner lipid layer 200. In some embodiments, the photosensitizer 500 is attached to the inner lipid layer 200.

In still another embodiment, a medical nanoparticle comprising the aforementioned first nanoparticle and the second nanoparticle described above.

In still another embodiment, referring to FIG. 3C, a medical nanoparticle comprising a core 100, an outer lipid layer 300, a inner lipid layer 200, a photosensitizer 500, and a nucleic acid 400. The core comprises a biodegradable ion precipitate 110. The inner lipid layer 200 is located between the core 100 and the outer lipid layer 300, and the photosensitizer 500 is located on the surface of the inner lipid layer 200 between the outer lipid layer 300 and the inner lipid layer 200. The nucleic acid 400 is located on the surface of the core 100. In some embodiments, the inner lipid layer 200 can be the surface of the core 100 and the nucleic acid 400 is embedded on the inner lipid layer 200. In other words, nucleic acid 400 One end is located inside the core 100 and the other end is located outside the core 100. In some embodiments, the photosensitizer 500 is attached to the inner lipid layer 200.

Wherein, the nucleic acid 400 in any of the above embodiments has the ability to inhibit the expression and action of an epidermal growth factor receptor (EGFR). This epidermal growth factor receptor is a cell surface receptor. When the epidermal growth factor receptor binds to epidermal growth factor (EGF), it activates the activation of various downstream kinases (such as PKC, ERK, AKT, and JAK), which in turn promotes cell growth and inhibits its death. Moreover, this epidermal growth factor receptor is known to be excessively expressed on a variety of cancer cells and participate in the canceration reaction therein. Based on this performance characteristic, a nucleic acid according to any of the embodiments of the present invention, a medical nanoparticle having a nucleic acid according to any of the embodiments of the present invention, or a pharmaceutical composition having a nucleic acid according to any of the embodiments of the present invention may utilize nucleic acid 400 (for example, ribonucleic acid interference such as small interfering ribonucleic acid, small hairpin ribonucleic acid or microribonucleic acid) to inhibit the expression and action of epidermal growth factor receptors, thereby inhibiting the growth of cancer cells and/or promoting cancer cell death.

Wherein, the photosensitizer 500 in any of the foregoing embodiments releases free radicals after being irradiated/excited by a specific light source, thereby causing oxidative damage to the cell target (for example, cell membrane, organelle, enzyme or DNA), thereby achieving tumor formation. treatment effect. In addition, the photosensitizer 500 emits fluorescence after being irradiated/excited by a specific light source, thereby detecting cancer by detecting fluorescence. In addition, it is also possible to pass through a dye which emits visible/invisible light after receiving/exciting a specific light source, thereby performing diagnosis of cancer by detecting visible/invisible light. For example, the light emitted by the dye may be fluorescent, luminescent, ultraviolet, visible or infrared light, as long as the light emitted by the dye can be detected by the photodetector.

Furthermore, the medical nanoparticle in any of the foregoing embodiments can be enhanced by retention The enhanced permeability and retention effect (EPR effect) enters and stays in cancer cells to produce a poisoning/inhibiting effect. In general, most solid tumors have (1) higher vascular density than normal tissues or organs; (2) structurally defective blood vessels (eg, large gaps between endothelial cells or lack of smooth muscle layer, etc.) And; (3) incomplete development of the lymphatic system; such characteristics can cause specific molecules to enter and accumulate in solid tumors on the one hand, and on the other hand make these specific molecules difficult to be cleared by related immune cells, resulting in the so-called Retention enhancement effect. Therefore, medical nanoparticles can utilize the retention enhancement effect to specifically target specific cancer cells. Among them, the cancer cells may be derived from prostate cancer, lung cancer, breast cancer, melanoma skin cancer, blood cancer, pancreatic cancer, ovarian cancer, liver cancer, colorectal cancer, neuroblastoma, glioblastoma, head and neck cancer or oral cavity. Cancer cells such as cancer.

Moreover, in some embodiments, the simultaneous presence of nucleic acid 400 and photosensitizer 500 can treat tumors using different inhibition pathways, respectively, to achieve a better therapeutic effect.

In some embodiments, the nucleic acid 400 of any of the preceding embodiments can comprise a nucleotide sequence of SEQ ID NO: 1. In other embodiments, nucleic acid 400 can comprise a nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the nucleic acid 400 of any of the foregoing embodiments can be ribonucleic acid interference (RNAi).

In some embodiments, the ribonucleic acid interference can be small interference ribonucleic acids (siRNA). Wherein, the small interfering ribonucleic acid can generally be in the form of a double strand having a smooth end (blunt), 3'-overhang (3'-overhang) or 5'-overhang (5'-overhang). In some embodiments, the small interfering ribonucleic acid has a smooth terminus and its positive strand comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In other embodiments, the ribonucleic acid interference can be small hairpin ribonucleic acid (shRNA). Wherein, the small hairpin ribonucleic acid is connected to the positive and negative strands by a spacer formed by a small length of nucleic acid, thereby forming a loop structure. In some embodiments, the positive strand of the small hairpin ribonucleic acid comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In still other embodiments, the ribonucleic acid interference can be provided in the form of micro-ribonucleic acids (miRNAs) or precursors thereof (eg, pri-miRNA or pre-miRNA). In some embodiments, the microribonucleic acid or precursor thereof comprises a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the photosensitizer 500 of any of the foregoing embodiments is selected from the group consisting of pyropheophorbide-phosphatidic acid (Pyro-PA), porphyrins. Photosan, photofrin (PH), tin etiopurpurin (SnET2), benzoporphyrin derivative (BPD) and 5-aminolaevulinic acid (5-aminolaevulinic acid, ALA).

Among them, pyropheophorbide is a chlorophyll derivative, and pyropheophorbide-phosphatidic acid is composed of pyropheophorbide and 1-palmitoquinone-2-hydroxy-sn-glycerol-3. - a lipid derivative formed by the reaction of 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphocholine. The pyropheophorbide-phosphatidic acid coated in the medical nanoparticle has a more significant biological activity than the pyropheophorbide-phosphatidic acid present alone. A light source suitable for exciting medical nanoparticle containing pyropheophorbide-phosphatidic acid is mainly a wavelength between 400 and 1,000 The source of nanometer (nm). Light sources of different wavelengths can be used to meet different needs. For example, in the treatment of superficial cancer, blue, green or yellow light with a wavelength between 400-600 nm can be used as the excitation source; in the treatment of deeper cancer, the wavelength can be used at 600. -1,000 red or near-infrared light as excitation light. In a particular embodiment, a light source having a wavelength of 410 nm can be utilized to excite medical nanoparticle comprising pyropheophorbide-phosphatidic acid to generate free radicals for poisoning.

In some embodiments, the biodegradable ion precipitate is selected from the group consisting of calcium phosphate (CaP), calcium citrate, calcium carbonate, magnesium carbonate, magnesium phosphate, and manganese phosphate. For example, suppose the biodegradable ion precipitate is calcium phosphate. In preparation, calcium chloride (CaCl ₂ ), sodium hydrogen phosphate (Na ₂ HPO ₄ ), and nucleic acid (if There is a uniform mixing, which is coated with the characteristics of mutual attraction between positive and negative charges, thereby forming a core comprising calcium phosphate and nucleic acid.

In some embodiments, the inner lipid layer and the outer lipid layer constitute a lipid bilayer structure, thereby increasing the stability of the medical nanoparticle in solution and the absorption of the medical nanoparticle by the biological cell.

In some embodiments, the inner lipid layer may coat the core and bond to the core using the properties of a combination of a cation and an anion.

In some embodiments, the inner lipid layer can be an anionic lipid layer and the outer lipid layer can be a cationic lipid layer. Among them, the cationic lipid layer is coated on the periphery of the anionic lipid layer by utilizing the property of combining a cation and an anion.

In other embodiments, the inner lipid layer can be an anionic lipid layer, while the outer fat The layer can be a neutral lipid layer. Among them, the neutral lipid layer is coated on the periphery of the anionic lipid layer by utilizing the characteristics that the hydrophobic ends of the lipids aggregate with each other.

In some embodiments, the anionic lipid layer can include, but is not limited to, dioleoyl phosphatidic acid (DOPA), 2,3-dihydrothio-1-propanesulfonic acid (2,3-dimercapto-1) -propanesulfonic acid, DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), diolein phospholipid 醯 serine ( Dioleoyl phosphatidylserine, DOPS), palmitoyl oleoyl phosphatidylserine (POPS), 1,2-dimyristyl-sn-glycerol-3-phosphoglycerol (1,2-dimyristoyl- Sn-glycero-3-phosphoglycerol, DMPG), 1,2-dipalmito-sn-glycero-3-phosphoglycerol (DPPG), 1,2-di 1,2-dioleoyl glycero-3-phospho-1-glycerol (DOPG), palmitoyl oleoyl phosphatidylglycerol (POPG), dimyristoyl phosphatidic acid Dimyristoyl phosphatidic acid, DMPA), dipalmitoyl phosphatidic acid (DPPA), dioleoyl phosphatidic acid (DOPA), palm oil phosphatidic acid (palmitoyl oleoyl p Hosphatidic acid, POPA), cholesterol cholesteryl hemisuccinate (CHEMS) or a derivative thereof.

In some embodiments, the cationic lipid layer can include, but is not limited to, 1,2-dioleoyl-3-trimethylammonium-propane chloride salt, DOTAP), dimyristoyl trimethylammonium propane, DMTAP), 1,2-dipalmitoyl-3-trimethylammonium propane (DPTAP), deacylated phosphatidylinositol manno-oligosaccharides (dPIMs), 1,2-dioleoyl oxypropyl-3-dimethyl hydroxyethyl ammonium bromide (DORIE), dimethyl dioctadecylammonium Bromide, DAAB), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-diolean-sn-glycerol-3-ethyl Phosphocholine (1,2-dioleoyl-sn-glycero-3-ethyl phosphocholine, DOEPC) or a derivative thereof.

In some embodiments, the neutral lipid layer can include, but is not limited to, dioleoyl phosphatidylcholine (DOPC), 1-palmitin-2-oleyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) , DOPE) or a derivative thereof.

Continuing the above examples, it is assumed that the anionic lipid uses dioleic acid phosphatidic acid and the cationic lipid uses 1,2-dioleyl-3-trimethylammonium-propane chloride salt, and at the time of preparation, the core containing calcium phosphate and nucleic acid is The bismuth phosphatidic acid is mixed; wherein the calcium ions in the core react with the phosphate of the diterpenoid phosphatidic acid, so that the diterpenoid phosphatidic acid is coated on the periphery of the core to form a linoleic layer. Next, the cationic lipid 1,2-dioleyl-3-trimethylammonium-propane chloride salt is added, and the 1,2-dioleoyl-3-trimethyl group is also reacted by the reaction of anion and cation. The ammonium-propane chloride salt is coated on the periphery of the inner lipid layer to form an outer lipid layer.

In certain embodiments, please refer to Figures 3A through 3C, further comprising a lipid-polyethylene glycol conjugate 310, and the lipid-polyethylene glycol conjugate 310 is linked externally Grease layer 300.

Lipid-polyethylene glycol conjugate 310 includes lipids and polyethylene glycols. Among them, the lipid system is used to bond the polyethylene glycol and the outer lipid layer 300. Polyethylene glycol is a polymer that increases the liposome circulation lifetime, which is commonly used to attach specific molecules (eg, antibodies, drugs, protein peptides or ligands) to the surface of liposomes. In addition, since polyethylene glycol is a polymer having a certain molecular length, it has a high degree of freedom, so that it can effectively shield medical nanoparticles from being attacked by macrophages or white blood cells. In the process, polyethylene glycol can be first linked to lipids, and then specific molecules (ie, antibodies, drugs, protein peptides or ligands, etc.) are added to form a "lipid-polyethylene glycol-specific molecule" conjugate. Next, in the preparation of the outer lipid layer, a "lipid-polyethylene glycol-specific molecule" conjugate is added, and the lipid in the conjugate uses its hydrophobic end and the lipid hydrophobic end of the outer lipid layer of the liposome. Aggregation further stabilizes the conjugate into the outer lipid layer.

In some embodiments, a particular molecule can be a target substance to increase the specificity of cancer cells. In some embodiments, the target substance can be a-Enolase. In some embodiments, the target material can be a benzamide derivative. In some embodiments, as shown in Figures 3A through 3C, the target material can be anisamide (AA) 320. For example, when preparing the outer lipid layer, the 1,2-dioleyl-3-trimethylammonium-propane chloride salt and the lipid-polyethylene glycol-anechoguanamine conjugate are simultaneously added, thereby The fennel amine is attached to the surface of the medical nanoparticle.

Because fennelamine binds to sigma receptors, The sigma receptor is a molecule that overexpresses the surface of cancer cells. Therefore, fennelamine can be used as a target for labeling cancer cells. Accordingly, by using this binding property, anisamine is attached to the surface of the medical nanoparticle of any of the examples, thereby increasing the specificity and therapeutic effect of the medical nanoparticle on cancer cells. Furthermore, based on the samarium receptor labeled to the surface of cancer cells, the medical nanoparticle with fennel amine on the surface can be specifically labeled to cancer cells without normal cells other than cancer cells. The cells produce a poisoning/inhibiting effect to enhance the therapeutic effect.

Among them, the lipid suitable for linking polyethylene glycol may include, but is not limited to, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine, DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dilaudium-sn-glycerol- 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), cholesterol (cholesterol) or oleic acid.

In other embodiments, the outer lipid layer may further comprise a cholesterol. Cholesterol is one of the elements forming a cell membrane and a liposome, and has an action of regulating membrane fluidity, and is therefore also called a fluidity buffer for liposomes; adding cholesterol to a chemical reaction increases the stability of the liposome. Therefore, in this embodiment, 1,2-dioleyl-3-trimethylammonium-propane chloride salt, lipid-polyethylene glycol-anesylamine conjugate and cholesterol are simultaneously added. These components are collectively expressed in the lipid layer other than the medical nanoparticle.

In some embodiments, the medical nanoparticle has a specific amount of inclusions (ie, A specific amount of nucleic acid and photosensitizer) to balance the charge and stabilize the molding. In particular, where applicable, medical nanoparticles include nucleic acids and photosensitizers in specific ratios/concentrations to produce a relatively preferred therapeutic effect. In certain embodiments, the weight ratio (micrograms per microgram) of nucleic acid and photosensitizer in the medical nanoparticle is between about 1:1 and 1:32. Wherein, the concentration of the nucleic acid may be about 1-10 mg per ml, and the concentration of the photosensitizer may be about 10-50 mg per ml. In a particular embodiment, the weight ratio (micrograms per microgram) of nucleic acid and photosensitizer in the medical nanoparticle is about 1:16. Wherein, the concentration of the nucleic acid may be about 2 mg per ml, and the concentration of the photosensitizer may be about 32 mg per ml.

In some embodiments, the core of the medical nanoparticle (having a linoleic layer) having a nucleic acid and a photosensitizer has an average diameter of from about 5 to about 50 nanometers. For example, the diameter of the core can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 Nano. In the preferred case, the core has an average diameter of about 5-30 nm. In the better case, the core has an average diameter of about 6-20 nm.

In some embodiments, after coating the core with a lipid bilayer structure (ie, an inner and outer lipid layer), the medical nanoparticles have an average diameter of about 12-50 nm; that is, the diameter of the medical nanoparticle. Can be 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nm. In preferred cases, the medical nanoparticles have an average diameter of from about 15 to about 20 nanometers. In a better case, the medical nanoparticles have an average diameter of about 20 nm.

In some embodiments, after coating the core with a lipid bilayer structure, the surface potential of the medical nanoparticle is about 10-70 millivolts (millivolt, mV); that is, the surface potential of the medical nanoparticle can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 millivolts. In a particular embodiment, the surface potential of the medical nanoparticle is about 20-60 millivolts.

Further, the nucleic acid or medical nanoparticle of any of the foregoing embodiments can be further prepared into a pharmaceutical composition. In other words, the pharmaceutical composition comprises a pharmaceutically acceptable additive and the nucleic acid or medical nanoparticle of any of the preceding embodiments.

Herein, the preparation of each pharmaceutical composition is in accordance with an acceptable, established medical procedure. The choice of a pharmaceutically acceptable additive for use with a nucleic acid, medical nanoparticle is substantially dependent on the desired form of the product of the pharmaceutical composition. The additive may be a diluent, an excipient, a disintegrants, a granulation binder, a lubricant, a filler, a flavoring agent, a coloring matter, an emulsifier, a suspending agent, or a fatty acid. , oils, dispersing agents, surfactants, bioavailability enhancers, or combinations thereof.

In some embodiments, the aforementioned pharmaceutical compositions can be administered by any suitable route, for example, by oral, parenteral (e.g., intramuscular, intravenous, subcutaneous or intraperitoneal injection), topical or transmucosal administration.

The nucleic acid or medical nanoparticle of any of the foregoing embodiments may be used if taken orally. Formulated as a lozenge with different additives. Among them, the additive may include an excipient, a decomposing agent, and a granulated binder. Here, suitable excipients may be, for example, microcrystalline cellulose, sodium citrate, calcium carbonate, dicalcium phosphate or glycine; Suitable decomposers may be, for example, starch, alginic acid and certain niobates; and suitable granulated binders may, for example, be polyvinyl pyrrolidone, sucrose, gelatin and acacia. Further, a lubricant such as magnesium stearate, sodium lauryl sulfate or talc may be added.

In addition, the nucleic acid or medical nanoparticle of any of the above embodiments may be formulated together with different additives to form a gelatin capsule. Herein, a solid composition can also be placed in the gelatin capsule as a filler. Among them, suitable fillers may be, for example, lactose, high molecular weight polyethylene glycol or a combination thereof.

When oral administration of liquid suspensions and/or elixirs is desired, the nucleic acid or medical nanoparticles can be combined with different flavoring agents, colorants or pigments, and if desired, emulsifiers and/or suspending agents, and A diluent such as water, ethanol, propylene glycol, glycerin, and combinations thereof is added.

The nucleic acid or medical nanoparticle of any of the above embodiments may be disposed in a liquid pharmaceutical composition if it is administered parenterally, and it may be a sterile solution or suspension. For example, a liquid pharmaceutical composition can be administered by intravenous, intramuscular, subcutaneous or intraperitoneal injection. Suitable diluents for the administration of sterile injectable solutions or suspensions include, but are not limited to, 1,3-butanediol, mannitol, water, Ringer's solution, and Isotonic sodium chloride solution. Preparation can be injected The ejaculation can also use fatty acids (such as oleic acid and its glyceride derivatives) or pharmaceutically acceptable oils in nature (such as olive oil or castor oil). )). Such oil solutions or suspensions may also contain an ethanol diluent or carboxymethyl cellulose or similar dispersing agents. For the preparation, commonly used surfactants (such as Tweens, polysorbate (Spans)), other similar emulsifiers or commonly used for medicinal use can also be used. Accepting bioavailability enhancers of the dosage form.

If a topical surface application route is employed, the nucleic acid or medical nanoparticle can be formulated with a variety of cutaneously acceptable inert excipients for a variety of dosage forms suitable for topical application. The topical surface-coated pharmaceutical composition can be applied in the form of a liquid, a paste, a lotion, an ointment, a gel, a spray, an aerosol, a skin patch, and the like. Typical inert excipients can be water, ethanol, polyvinyl pyrrolidone, propylene glycol, mineral oil, stearyl alcohol or gel-forming materials.

If the transmucosal absorption pathway is employed, the nucleic acid or medical nanoparticle can be formulated with various biodegradable polymeric excipients into various mucosally absorbable dosage forms; for example, the buccal and/or sublingual delivery of the drug through the oral mucosa Drug dosage unit. Wherein, various pharmaceutically acceptable biodegradable polymeric excipients can be used to provide a suitable degree of adhesion and a desired drug release profile, and which can be present in the cheek and/or with the active agent being administered and/or Or the sublingual drug dosage unit is compatible with the ingredients. Typically, the polymeric excipient comprises a hydrophilic polymer attached to the wet surface of the oral mucosa. Examples of polymeric excipients include, but are not limited to, acrylic acid polymers and copolymers, hydrolyzed hydrolyzed Polyvinylalcohol), polyethylene oxides, polyacrylates, vinyl polymers and copolymers, polyvinylpyrrolidine, dextran, guar gum, pectin, starch and cellulose polymer.

Depending on the actual needs, the pharmaceutical composition may be in the form of a solid, semi-solid, liquid, cream, capsule, spray or patch. Accordingly, the pharmaceutical composition can be administered orally, parenterally (e.g., intramuscularly, intravenously, subcutaneously or intraperitoneally), topically or transmucosally, to produce optimal therapeutic effects for different clinical conditions.

After administration to an individual, the pharmaceutical composition reaches the tumor site via the systemic circulation and is labeled to cancer cells and/or cancer by the retention effect of medical nanoparticles or its fennel amine (or other target substance). Organization. Thereafter, the medical nanoparticle can be efficiently fused to the cell membrane and absorbed into the cancer cells/tissue. Once inside the cell/tissue, the medical nanoparticle releases the EGFR siRNA, which in turn blocks the expression of epidermal growth factor, thereby inhibiting cancer cell growth and/or promoting its death. At the same time, if a suitable illumination is applied, the pyropheophorbide-phosphatidic acid in the nanoparticles can be activated to generate free radicals to cause oxidative damage to the cancer cells/tissue.

As previously mentioned, in collocation applications, a source having a wavelength between 400 and 1,000 nm can be utilized to stimulate activation of pyropheophorbide-phosphatidic acid in medical nanoparticles. Different wavelengths have different excitation energies, and the type/location/characteristic/size of the cancer to be applied varies. In one embodiment, a source having a wavelength of 410 nm is used to excite pyropheophorbide-phosphatidic acid in medical nanoparticles.

In the following, a number of experimental examples are presented to illustrate certain aspects of the invention, so that those skilled in the art to which the invention pertains can practice the invention and should not To the extent that the scope of the invention is limited. It is believed that one skilled in the art can, after having the benefit of All publications cited herein are hereby incorporated by reference in their entirety.

Oral cancer cell culture

The human oral cancer cells SCC4 and SAS used in the experimental examples were cultured in DMEM cell culture medium containing 10% fetal bovine serum (FBS). The cells were cultured in an environment containing 5% carbon dioxide at 37 ° C until a sufficient number of cells were cultured for subsequent analysis.

Preparation of Nanoparticles Coated with EGFR siRNA

Lipid calcium phosphate (LCP) was used to coat the siRNA. The principle is to form a nano core of Calcium phosphate (CaP) by microemulsion, and then coated with a layer of cationic lipid 1,2-dioleyl-3-trimethylammonium-propane chloride (1,2- Dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) and polyethylene glycol PEG (polyethylene glycol, PEG), and can also modify anisamide (AA) on PEG. Anisamide can be effectively adsorbed on the sigma receptor on the cancer cell membrane to achieve the target. The preparation method is as follows. CaCl ₂ and Na ₂ HPO _{4 are} added to the oil phase (cyclohexane/lgepal CO-520) and anionic lipid diol phosphatidic acid (DOPA) and EGFR siRNA are added to form Calcium phosphate (CaP). ) Nano core. Impurities were added by adding absolute alcohol (absolute Ethanol) and centrifuged at 12,500 rpm for 10 min. Further, chloroform was added and centrifuged at 10,000 rpm, and the supernatant was taken as the LCP core. The LCP core is further added with DOTAP, cholesterol, 1,2-distearate-sn-glycero-3-phosphoethanolamine-polyethylene glycol conjugate, optionally with or without the target substance anisamide. The nitrogen was blown dry and evacuated under vacuum to complete the finished product.

Preparation of nanoparticle coated with photosensitizer Pyropheophorbide

Attaching a phosphatidic acid to pyropheophorbide to pyropheophorbide-phosphatidic acid, which has hydrophilic and hydrophobic properties for chimeric into the inner layer of lipid calcium phosphate The fluid is set with phospholipids. The preparation process is based on pyropheophorbide as a photosensitizer, which can photochemically react with light of a specific wavelength, so it needs to be protected from light throughout the process. The preparation method is as follows: CaCl ₂ and Na ₂ HPO _{4 are} added to the oil phase (cyclohexane/lgepal CO-520) and DOPA and pyrofophorbide-phosphatidic acid are added to form a Calcium phosphate (CaP) nano core. Impurities were screened out by adding absolute ethanol and centrifuged at 12,500 rpm for 10 min. Further, chloroform was added and centrifuged at 10,000 rpm, and the supernatant was taken as the LCP core. The LCP core is further added with DOTAP, Cholesterol, DSPE-PEG, optionally with or without the target material anisamide, and then dried with nitrogen, and vacuum evacuated to complete the finished product.

Cytotoxicity test (MTT test)

MTT reagent (ie 3-(4,5-dimethylthiazol)-2,5-diphenyltetrazoliumbromide) is a yellow dye with succinate dehydrogenase-ubiquinone (SDH) and cytochrome C (cytochrome C) After the action, a blue-violet crystal which is insoluble in water is formed. After dissolving the crystal in a DMSO solution, the cell survival condition can be judged by using the absorbance of the solution at 570 nm, thereby converting the survival ratio of the cells.

After preparing 5 mg of MTT solution per ml in DMEM medium containing no serum, the solution was filtered through a filter membrane having a pore size of 0.22 μm, and the resulting filtrate was used for analysis and subsequent cell test.

SCC4 or SAS cells were seeded in 48 wells (1 × 10 ⁴ cells per well), treated with porphyrin photophotos (photosan) and pyropheophorbide-phosphatidic acid for 48 hours, respectively. The cells were washed with 100 μl of physiological saline, and then 20 μl of MTT solution was added. After standing at 37 ° C in a cell incubator containing 5% carbon dioxide for 4 hours, the MTT solution was removed and 300 μl of DMSO was added. Allow to stand at room temperature for 30 minutes in the dark. 200 microliters of the supernatant taken to a 96-well plate, using the enzyme linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA ) reading machine ^{(VersaMax TM Microplate Reader, Molecular devices} , Sunnyvale, USA) in the supernatant was read The absorbance at 570 nm.

Nanoparticle analysis

One microgram of nanoparticle was dropped onto a carbon-coated copper mesh, and dried under vacuum for 2 days, and then delivered to a large-scale biotransparent and scanning electron microscope (Hitachi, HT7700, Japan).

In vivo imaging system (IVIS) analysis

The first set of experiments can observe the metastatic condition of cancer cells. In the first set of experiments, the firefly was used to first transfer the gene encoding the luciferase to cancer cells, and after screening with antibiotics, cells with stable luciferase gene expression were selected. The cancer cells with stable luciferase gene expression were then inoculated subcutaneously into the right thigh of the mouse. When the tumor was as long as about 200 mm 3 , the mice were grouped (including the administration group and the non-administration group, and different groups according to the experimental design), and the treatment was performed. The cancer cells were then imaged and recorded in a non-invasive live imaging system (IVIS Imaging System, Lumina series III).

In the second set of experiments, the transmission of photosensitizer-containing nanoparticles in mice was observed. The second set of experiments was performed by injecting nanoparticles (2 mg of EGFR siRNA per ml and 16 mg of photosensitizer nanoparticle per ml/200 μl per ml) from the tail vein. The mice were placed in the dark for 55 minutes. Before entering the instrument, the rats were temporarily paralyzed with a gastrodia elata, and then the mice were irradiated with a 660 nm source in a non-invasive living molecular imaging system to excite the pyropheophorbide-phosphatidic acid in the nanoparticles, and then photographed separately. Photographic measurements were taken for absorbance values in the 670-690 nm and 710-730 nm wavelength sections.

Animal test

Human oral cancer cells SCC4 (6x10 ⁵ ) were implanted into the subcutaneous tissue of mice (BALB/cAnN.Cg-foxnlnu/CrlNarl, 8 weeks old). After the tumor grew to 200 mm 3 , nanoparticles (15-45 mM / 200 μl) were injected from the tail vein. The mice were placed in the dark for 55 minutes, after which the mice were irradiated with a 410 nm light source to excite the pyropheophorbide-phosphatidic acid in the nanoparticles, and then measured at 670-690 nm and The absorbance of the 710-730 nm wavelength section.

On day 0, human oral cancer cells SAS (6x10 ⁵ ) were planted in subcutaneous tissue of mice (BALB/cAnN.Cg-foxnlnu/CrlNarl, 8 weeks old). After the tumor grew to 200 mm 3 , a dose of nanoparticle (15-45 mM / 200 μl) was injected from the tail vein for a total of three doses. After the third dose, the mice were placed in the dark for 55 minutes, then the mice were fixed with a Baoding device, and photodynamic therapy was performed using a plant light source (the wavelength of which can be visible light). The total energy of the light was per square centimeter. 100 joules. Thereafter, changes in tumor size and body weight of the mice were measured daily.

Experimental Example 1 Poisoning effect of different kinds of photosensitizers

Figures 1A and 1B are graphs of cell viability obtained by MTT assay after treatment of SCC4 cells with different concentrations of porphyrin phototherapy and pyropheophorbide-phosphatidic acid; Concentration of 50% inhibition (IC ₅₀ ) of the two photosensitizers. The results showed that the semi-inhibitory concentration of porphyrin photoreceptors on SCC4 cells was 2.4 micrograms per milliliter; in contrast, the semi-inhibitory concentration of pyropheophorbide-phosphatidic acid on SCC4 cells was 0.08 micrograms per milliliter.

Similarly, after treatment of SAS cells with different concentrations of porphyrin phototherapy (Fig. 1C) and pyropheophorbide-phosphatidic acid (Fig. 1D), half of SAS cells can be obtained from porphyrin phototherapy. The inhibitory concentration was 1.75 micrograms per milliliter, and the semi-inhibitory concentration of pyropheophorbide-phosphatidic acid on SAS cells was 0.1 microgram per milliliter.

Figures 2A and 2B summarize the above results to more clearly show the difference in toxicity between the two photosensitizers. As can be seen from the figure, in SCC4 cells, the semi-inhibitory concentration of porphyrin phototherapy is 2.4 micrograms per milliliter, and the pyropheophorbide-phosphatidic acid is 0.08 micrograms per milliliter; the difference between the two is 30 times. As for the SAS cells, the semi-inhibitory concentration of the porphyrin phototherapy was 1.75 micrograms per milliliter, and the pyropheophorbide-phosphatidic acid was 0.1 microgram per milliliter; the difference was 17.5 times.

Accordingly, the experimental results confirmed that pyropheophorbide-phosphatidic acid has a better inhibitory effect on cancer cells than porphyrin phototherapy. The following experimental example will use pyropheophorbide-phosphatidic acid as a photosensitizer to prepare nanoparticles and perform correlation analysis.

Experimental Example 2 Optimal coating amount of nano particles

After confirming the use of pyropheophorbide-phosphatidic acid as a photosensitizer, Experimental Example 2 will further analyze the optimum of EGFR siRNA and pyropheophorbide-phosphatidic acid in nanoparticles. content.

As shown in Figures 4A to 4C, nanoparticle coated with 2 mg of EGFR siRNA per ml and 32 mg of photosensitizer per ml did not stably form spherical particles (Fig. 4A). In contrast, 2 mg of EGFR siRNA per ml and 16 mg of photosensitizer nanoparticle per ml (Figure 4B), and 1 mg of EGFR siRNA per ml and 16 mg of photosensitizer per ml The nanoparticles (Fig. 4C) are stable in the shape of the sphere.

From the experimental results, it is known that nanoparticle coated with 2 mg of EGFR siRNA per ml and 16 mg of photosensitizer per ml is the maximum amount that nanoparticle can coat. The following experimental example is a correlation analysis using nanoparticles containing this dose.

Experimental example 3 Particle size of nanoparticles

After preparing nanoparticles containing the largest amount of EGFR siRNA and pyropheophorbide-phosphatidic acid, the size of the nanoparticles was analyzed by a transmission electron microscope.

As shown in Figures 5A to 5F, the average particle size of the core (containing the lining layer) coated with EGFR siRNA was 11.1 ± 3.1 nm (Fig. 5A), and the average particle size of the nanoparticles coated with EGFR siRNA was 34.9±3.0 nm (Fig. 5B), the core of the coated pyropheophorbide-phosphatidic acid (containing the inner lipid layer) has an average particle size of 9-12 nm (Fig. 5C), coated with coke The average particle size of magnesium chlorophyllin-phosphatidic acid nanoparticles is 15-20 nm (Fig. 5D), and is coated with EGFR siRNA and the core of pyropheophorbide-phosphatidic acid (including the linoleic layer). The average particle size is 9-12 nm (Fig. 5E), while the average particle size of nanoparticles coated with EGFR siRNA and pyropheophorbide-phosphatidic acid is 20 nm (Fig. 5F) .

Experimental Example 4 Surface Potential of Nanoparticles

Preparation of the largest amount of EGFR siRNA and pyropheophorbide-phospholipid After the acid nanoparticles, the surface potential of the nanoparticles was measured using a nanoparticle size analyzer (Zetasizer Nano S90, Malvern, Zurich, Switzerland) to analyze the relevant physical properties.

As shown in Table 1, the surface potential of the nanoparticles containing no fennel amine averaged 25.0 ± 0.5 mV; in comparison, the surface potential of the nanoparticles containing fennel amide was 45.4 ± 4.5 on average. millivolt.

^a mean ± standard deviation (quantity is 3)

The results show that the addition of fennelamine increases the surface potential of the nanoparticles; that is, fennel amide can provide higher stability and dispersibility of the nanoparticles.

Experimental Example 5 Analysis of the target nanoparticle to the tumor site and time required

As mentioned above, the nanoparticles are excited by a light wave of 410 nm, and the divergent light is detected in the wavelength sections of 670-690 nm and 710-730 nm. Figure 6 shows the photon values of the two bands (the 670-690 nm mark is red line and the 710-730 nm mark is blue line), and the time at which the nanoparticles arrive at the tumor site is analyzed. The results showed that no matter which band was used to measure the photon value of the tumor site, it was found that the nanoparticles began to aggregate at 5 minutes after the injection and reached the maximum amount after 55 minutes.

The results clearly indicate that the nanoparticles can be accurately labeled to the tumor and will reach the highest amount 55 minutes after injection.

Experimental Example 6 Treatment of Tumors Using Nanoparticles

It can be seen from Experimental Example 5 that after intravenous injection, the nanoparticles aggregated at the tumor site and reached the maximum amount at 55 minutes. Therefore, Experimental Example 6 uses photodynamic therapy with 55 minutes as the interval time.

Figure 7 shows the daily tumor volume change of nude mice xenografted with SCC4 oral cancer cells on days 0-13. △ represents LCP-EGFR siRNA or LCP-Control siRNA or phosphate buffered saline in the tail vein; ▲ represents LCP-Pyro-PA in the tail vein or not. The in vivo animal experiment is based on the xenograft human oral cancer SCC4 nude mouse model validation combination therapy, using BALB/c nude mice subcutaneous transplantation of oral cancer cells SCC4, and waiting for the tumor to grow to 200mm ³ to five groups of experiments, respectively A : control group (PBS (phosphate buffered saline)), B: photodynamic control group (LCP-Control siRNA + photodynamic reagent (PDT)) and C, D: EGFR siRNA control group (divided into LCP-EGFR siRNA + LCP) -Pyro-PA unilluminated group and LCP-EGFR siRNA + PBS concomitant light group) and E: combination treatment group (LCP-EGFR siRNA + PDT), tumor growth was observed during the 14 consecutive days of the experiment.

On day 14, A group SCC4 xenograft tumor volume in nude mice of 782.6mm ^3; B Group Tumor volume was 462.2mm ^3, group A reduction in tumor volume than 320.4mm ^3, can be estimated PDT tumor volume inhibition ratio of 40.9%; group C is the tumor volume 388.7mm ^3, 393.9mm ³ than in group A decrease in tumor volume, inhibiting tumor volume ratio of 50.3%; D group tumor volume was 381.8mm ^3, 400.8mm ³ than in group A decrease in tumor volume, inhibiting tumor volume ratio was 51.2% (p <0.001); E tumor volume was 155.6mm ^3, 627.0mm ³ to reduce tumor volume than in group a, the combination treatment of tumor inhibition was 80.1%. Group E was the best to suppress tumor volume compared to other treatments alone (EGFR siRNA or PDT).

Therefore, it can be seen from the results that the nanoparticle having the EGFR siRNA itself has the effect of inhibiting tumor growth; if photodynamic therapy is performed with an appropriate illumination, a better therapeutic effect can be achieved.

In summary, in some embodiments, nucleic acids, medical nanoparticles, and pharmaceutical compositions according to the present invention may utilize nucleic acids (eg, ribonucleic acid interference such as small interfering ribonucleic acid, small hairpin ribonucleic acid, or microribonucleic acid). To inhibit the expression and action of the epidermal growth factor receptor, thereby inhibiting the growth of cancer cells and/or promoting cancer cell death. In some embodiments, the photosensitizer in the medical nanoparticle releases free radicals upon exposure/excitation of a particular source of light, thereby causing oxidative damage to the cancer cells. In some embodiments, the photosensitizer in the medical nanoparticle is more useful for diagnosis. In some embodiments, the nucleic acid, medical nanoparticle, and pharmaceutical composition according to the present invention can simultaneously treat tumors with different inhibition pathways using nucleic acids and photosensitizers in order to achieve better therapeutic effects.

The specific embodiments of the present invention are disclosed in the above embodiments, and are not intended to limit the present invention, and those skilled in the art without departing from the spirit and scope of the invention Various changes and modifications may be made thereto, and the scope of the invention is defined by the scope of the appended claims.

<110> Chung Yuan University

<120> Nucleic acid, medical nanoparticle group, and pharmaceutical composition

<130> N/A

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 25

<212> RNA

<213> Artificial Sequence

<220>

<223> EGFR siRNA

<400> 1

<210> 2

<211> 21

<212> RNA

<213> Artificial Sequence

<220>

<223> EGFR siRNA

<400> 2

100‧‧‧ core

110‧‧‧Biodegradable ionic precipitates

500‧‧‧Photosensitizer

200‧‧‧ Inner fat layer

300‧‧‧ outer lipid layer

310‧‧‧Lipid-polyethylene glycol conjugate

320‧‧‧Fennelamide

Claims (14)

A medical nanoparticle group comprising: a first core comprising a biodegradable ion precipitate; a first outer lipid layer; a first inner lipid layer located at the first core and the first outer lipid layer And a nucleic acid located on the surface of the first core, the nucleic acid being the nucleotide sequence of SEQ ID NO: 1. The medical nanoparticle group according to claim 1, further comprising: a photosensitizer between the first outer lipid layer and the first inner lipid layer. The medical nanoparticle group according to claim 2, wherein the weight ratio of the nucleic acid to the photosensitizer is between 1:1 and 1:32. The medical nanoparticle group according to claim 1, wherein the first inner lipid layer is an anionic lipid layer, and the first outer lipid layer is a cationic lipid layer or a neutral lipid layer. The medical nanoparticle group according to any one of claims 1 to 4, further comprising at least one lipid-polyethylene glycol conjugate, the at least one lipid-polyethylene glycol conjugate being linked to the first An outer lipid layer. The medical nanoparticle group according to claim 5, further comprising at least one target substance, each of the target substances being linked to one of the at least one lipid-polyethylene glycol conjugate. A pharmaceutical composition comprising: a pharmaceutically acceptable additive; at least one of the medical nanoparticle groups of any one of claims 1 to 6 in the pharmaceutically acceptable additive; At least one other medical nanoparticle group is located in the pharmaceutically acceptable additive, each of the other medical nanoparticle groups comprising: a second core comprising a biodegradable ion precipitate (Bio- Degradable ionic precipitate, BIP); a second outer lipid layer; a second inner lipid layer between the second core and the second outer lipid layer; and a photosensitizer located in the second outer lipid layer Between the second inner lipid layers. The pharmaceutical composition according to claim 7, further comprising: another photosensitizer between the first outer lipid layer and the first inner lipid layer. The pharmaceutical composition according to claim 7, wherein the second inner lipid layer is an anionic lipid layer, and the second outer lipid layer is a cationic lipid layer or a neutral lipid layer. The pharmaceutical composition according to any one of claims 7 to 9, further comprising at least one lipid-polyethylene glycol conjugate, each of the lipid-polyethylene glycol conjugates being attached to the first outer lipid layer Or the second outer lipid layer. The pharmaceutical composition according to claim 10, further comprising at least one target substance, each of the target substance being linked to one of the at least one lipid-polyethylene glycol conjugate. A pharmaceutical composition comprising: at least one of the medical nanoparticle groups of any one of claims 1 to 6; and a pharmaceutically acceptable additive, the medical nanoparticle group being located in the pharmaceutical Acceptable additives. A nucleic acid which is a nucleotide sequence of SEQ ID NO: 1. A pharmaceutical composition comprising: the nucleic acid of claim 13; and a pharmaceutically acceptable additive, the nucleic acid being mixed with the pharmaceutically acceptable additive.